The purpose of the proposed studies is to determine how PAR1 and PAR4 interact to mediate thrombin signaling in platelets. Specifically, the project will identify the PAR1-PAR4 interaction interface and determine how the anionic region on PAR4 exodomain contributes to platelet activation in vivo in the presence of PAR1. The long-term goal is to identify potential targets for anti-platelet therapies that do not pose a risk for bleeding by understanding how PAR1 and PAR4 interact with one another to mediate thrombin signaling for platelet activation. Specific Aim 1 will characterize a blocking antibody to PAR4's anionic region and determine the role of this region in platelet activation in vivo. The proposed studies will first test the hypothesis that the anionic region on PAR4 is a potential target for anti-platelet therapy. An antibody to PAR4s anionic region, CAN12, blocks human and mouse platelet activation. Since CAN12 blocks 1-thrombin-induced human platelet aggregation, studies will determine its mechanism by examining influence of CAN12 on PAR1 activation when PAR1 is co- expressed with PAR4. In addition, studies will test the hypothesis that the anionic region of PAR4 is critical for PAR4 activation in vivo using transgenic animals expressing mouse PAR4 with mutations in the anionic cluster (mPAR4-AAA), mouse PAR1 or both under the control of the GPIb1 promoter. The transgenic animals will be compared in ex vivo platelet function assays, thrombosis assays and tail bleeding times. Specific Aim 2 will determine the importance of PAR1 and PAR4 interaction on the surface of cells for efficient activation by thrombin. We will determine the regions on PAR1 and PAR4 required for homodimer and heterodimer formation using Bioluminescence Resonance Energy Transfer (BRET) and cysteine crosslinking. The cysteine crosslinking studies will use an inducible system to examine homodimers and heterodimers over a wide range of expression levels to ensure that interactions are occurring in a physiological range and are not an artifact of overexpression. The BRET and crosslinking studies will be verified with bimolecular fluorescence complementation (BiFC) studies. Finally, we will test the hypothesis that PAR1 enhances PAR4 activation by physically interacting. The proposed studies will examine the interactions between PAR1 and PAR4 on the molecular level to determine how these receptors communicate with one another to mediate thrombin signaling in platelets. These studies have the potential to identify general mechanisms of GPCR interactions to mediate a range of signals that may be transferred to other GPCRs found on the platelet.